Compositions and methods for treating age-related diabetes and related disorders

ABSTRACT

The invention features compositions and methods treating or preventing for age-related insulin resistance, type 2 diabetes and related disorders. The method involves depleting fTreg cells with an anti-ST2 antibody to decrease age-related fTreg accumulation and restore insulin sensitivity, thereby treating age-related insulin resistance, type 2 diabetes and related disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 62/110,349, filed Jan. 30, 2015 and 62/154,652, filed Apr. 29,2015. The entire content of these applications are hereby incorporatedby reference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the following grants from the NationalInstitutes of Health, Grant Nos: 5 F30 DK096929-03; HL088093; andHL105278. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Aging and obesity are the two primary etiologies of insulin resistanceand diabetes, the defining epidemics of the modern world. While thecomplex interplay between the immune system and adipose tissueinflammation drives obesity-associated insulin resistance, theunderlying mechanisms of aging-associated insulin resistance have yet tobe determined. Age-associated insulin resistance is prevalent. In the USalone incidence of diabetes among adults, ages 65-79 years, hassignificantly increased from 6.9 per 1000 in 1980 to 15.4 per 1000 in2011. Over 25% of all Americans over 60 years have type 2 diabetes andmore than 50% are insulin resistant. Accordingly, methods of treatingage-related insulin resistance, type 2 diabetes and related disordersare urgently needed.

SUMMARY OF THE INVENTION

As described herein below, the present invention features compositionsand methods treating or preventing age-related insulin resistance, type2 diabetes and related disorders. The method involves depletingfat-resident regulatory T cells (fTregs) cells with an anti-ST2 antibodyto decrease age-related fTreg accumulation and restore insulinsensitivity, thereby treating age-related insulin resistance, type 2diabetes and related disorders.

In one aspect, the invention provides a method of enhancing insulinsensitivity in a cell or tissue, the method involving contacting thecell or tissue with an anti-ST2 antibody or antigen binding fragmentthereof, thereby decreasing the number of adipose-resident regulatory Tcells (fTreg) and enhancing insulin sensitivity relative to a reference.

In another aspect, the invention provides a method of reducing insulinresistance in a cell or tissue, the method involving contacting the cellor tissue with an anti-ST2 antibody or antigen binding fragment thereof,thereby decreasing the number of adipose-resident regulatory T cells(fTreg) and reducing insulin resistance relative to a reference. Invarious embodiments, the cell is an adipocyte. In various embodiments,the tissue is adipose tissue.

In yet another aspect, the invention provides a method of enhancinginsulin sensitivity in a subject, the method involving contacting thesubject with an anti-ST2 antibody or antigen binding fragment thereof,thereby decreasing the number of adipose-resident regulatory T cells inthe subject and enhancing insulin sensitivity relative to a reference.

In still another aspect, the invention provides a method of reducinginsulin resistance in a subject, the method involving contacting thesubject with an anti-ST2 antibody or antigen binding fragment thereof,thereby decreasing the number of adipose-resident regulatory T cells inthe subject and reducing insulin resistance relative to a reference.

In another aspect, the invention provides a method for treating orpreventing age-related insulin resistance, type 2 diabetes and relateddisorders in a subject, the method involving contacting the subject withan anti-ST2 antibody or antigen binding fragment thereof, therebytreating or preventing age-related insulin resistance, type 2 diabetesand related disorders in a subject.

In yet another aspect, the invention provides a method for treating orpreventing age-associated metabolic dysregulation in a subject, themethod involving contacting the subject with an anti-ST2 antibody orantigen binding fragment thereof, thereby treating or preventingage-associated metabolic dysregulation in the subject.

In one aspect, the invention provides a method for identifying a subjecthaving or at risk of developing age-related insulin resistance, type 2diabetes and related disorders, the method involving detecting anincrease in an ST2 polypeptide or polynucleotide expression in an fTregor detecting an increase in the number of fTregs relative to a control.In one embodiment the fTreg is present in a tissue biopsy. In anotherembodiment the tissue biopsy if obtained from visceral adipose tissue.In still another embodiment the ST2 polypeptide is detected in animmunoassay.

In another aspect, the invention provides a method of reducing insulinresistance in a subject, the method involving administering to thesubject an anti-ST2 antibody or antigen binding fragment thereof,wherein the subject is identified as having an increase in an ST2polypeptide or polynucleotide expression in an fTreg or detecting anincrease in the number of fTregs relative to a control. In oneembodiment the subject has or is at risk of developing age-relatedinsulin resistance.

In one aspect, the invention provides a kit for treating or preventingage-related insulin resistance, the kit containing an effective amountof an ST2 antibody in a pharmaceutical excipient.

In various embodiments of any of the previous aspects or any otheraspect of the invention delineated herein, the subject is fifty, sixty,or sixty-five, years old or older. In various embodiments of any of theprevious aspects or any other aspect of the invention delineated herein,the method reduces body weight relative to a reference. In variousembodiments of any of the previous aspects or any other aspect of theinvention delineated herein, the method decreased visceral adiposetissue and/or subcutaneous adipose tissue adiposity relative to areference. In various embodiments of any of the previous aspects or anyother aspect of the invention delineated herein, the method increasesrespiratory exchange ratio, oxygen consumption, and/or core bodytemperature relative to a reference.

In various embodiments of any of the previous aspects or any otheraspect of the invention delineated herein, the method reduces fastingserum glucose and insulin levels relative to a reference. In variousembodiments said method reduces glucose excursions during a glucosetolerance test and/or increases sensitivity during an insulin tolerancetest relative to a reference. In various embodiments said method reducesserum non-esterified free fatty acid (NEFA) levels relative to areference. In various embodiments said method increases insulinsensitivity relative to a reference. In various embodiments said methoddecreases hepatic steatosis relative to a reference. In variousembodiments said method decreases fasting hepatic and/or serumtriglyceride content.

In various embodiments of any of the previous aspects or any otheraspect of the invention delineated herein, the method does not producean autoimmune syndrome. In various embodiments said method specificallydecreases fTreg numbers while preserving splenic Treg numbers. Invarious embodiments said method increases the glucose uptake capacity ofvisceral adipose tissue compared to a reference. In various embodimentsof any of the previous aspects or any other aspect of the inventiondelineated herein, the anti-ST2 antibody administration is parenterally.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “ST2 (Interleukin 1 receptor-like 1) protein” is meant a proteinhaving at least about 85%, 90%, 95%, or 100% amino acid identity to NCBIAccession No. Q01638 or a fragment thereof that binds IL-33.

An exemplary amino acid sequence is provided below.

>sp|Q01638|ILRL1_HUMAN Interleukin-1 receptor-like 1 OS =Homo sapiens GN = IL1RL1 PE = 1 SV = 4MGFWILAILTILMYSTAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHSIYCIIAVCSVFLMLINVLVIILKMFWIEATLLWRDIAKPYKTRNDGKLYDAYVVYPRNYKSSTDGASRVEHFVHQILPDVLENKCGYTLCIYGRDMLPGEDVVTAVETNIRKSRRHIFILTPQITHNKEFAYEQEVALHCALIQNDAKVILIEMEALSELDMLQAEALQDSLQHLMKVQGTIKWREDHIANKRSLNSKFWKHVRYQMPVPSKIPRKASSLTP LAAQKQ

By “ST2 (Interleukin 1 receptor-like 1) antibody” is meant an antibodyor antigen binding fragment thereof that specifically binds an ST2polypeptide or fragment thereof. Such antibodies are known in the artand commercially available. A mouse anti-human ST2 antibody isavailable, for example, from Novus Biologicals. Other anti-human ST2antibodies are known in the art (e.g., Palmer et al., Arthritis Rheum.2009 March; 60(3):738-49).

By “fat-resident regulatory T cell (fTreg)” is meant a regulatory T cellthat is resident in adipose tissue. In one embodiment, an fTregexpresses Foxp3. In another embodiment, an fTreg expresses an increasedlevel of ST2 relative to a splenic Treg of fat conventional CD4⁺ Tcells. For example, ST2 is at least about 10, 20, 30, 40, 50, or 60times more highly expressed in an fTreg compared to a splenic Treg.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “alteration” is meant a change (increase or decrease) in the levelsor activity of an analyte detected by standard art known methods such asthose described herein. As used herein, an alteration includes a 10%change in analyte levels, preferably a 25% change, more preferably a 40%change, and most preferably a 50% or greater change in analyte levels.In one embodiment, an analyte is a cell, such as a fat regulatory Tcells (fTreg).

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

By “biopsy” is meant a test involving sampling of cells or tissues forexamination by removing tissue from a living subject to determine thepresence or extent of a disease. Examples of biopsies include removal ofvisceral adipose tissues or cells.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of or” consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include age-related insulin resistance, type 2diabetes and related disorders. Related disorders include metabolicdisorders characterized by an increase in the number of fTregs.

By “effective amount” is meant the amount of an agent of the invention(e.g., antibody or antigen binding fragment thereof) required toameliorate the symptoms of a disease relative to an untreated patient.In one embodiment, an effective amount is the amount required tosignificantly reduce the number of fat-resident regulatory T cells in aadipose tissue relative to the number present in an untreated control.The effective amount of active agent used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that can be subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition.

By “specifically binds” is meant a compound or antibody that recognizesand binds a polypeptide of the invention, but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are a schematic and pie charts showing that fTregs wereselectively enriched in aged mice. FIG. 1A is a schematic showing ptheetiologies for obesity-versus age-related insulin resistance in mice.FIG. 1B depicts pie charts showing visceral adipose tissue (VAT)adipo-immune profiles (AIP) from mice at 12 weeks (young, n=10), 44weeks (aged, n=10), and in diet-induced obese (DIO) mice (n=10). Immunecells abundance, expressed as percentage of CD45.2⁺ cells. FIG. 1C is abar graph comparing changes in immune cell abundance between indicatedgroups, expressed as fold change in cell number per gram of VAT. #,false discovery rate <2%. Obese mice were fed a high fat diet for 12weeks from 12 weeks of age. Data represents mean±standard error of themean (s.e.m).

FIGS. 2A-2C are 12 scatter plots showing selected gating strategies usedto generate adipolmmune Profiles (AIPs)—Adipolmmune Profiles weregenerated through the use of several distinct antibody cocktails. FIGS.2A-2C, using Foxp3^(Cre-YFP) reporter mice, show how the stromalvascular fraction of visceral adipose tissue (VAT) was analyzed by flowcytometry to identify several T cell subtypes (FIG. 2A), macrophagesubsets (FIG. 2B), and eosinophils and neutrophils (FIG. 2C).

FIGS. 3A-3H are scatter plots, linear, and bar graphs showing that fTregknock out (KO) mice were protected from general hallmarks of metabolicaging. FIG. 3A is a scatter plot showing representative FluorescenceActivated Cell Sorting (FACS) plots of fTreg KO (Foxp3^(Cre)PPARg^(fl/fl)) and control (Foxp3^(Cre) PPARg^(+/+)) mice depicting Tregenrichment in visceral adipose tissue (VAT) and spleen. FIG. 3B is a bargraph showing total body weight (n=15 per group), and lean and fat massof control and fTreg KO mice (˜12 months, n=8 per group). FIG. 3C is abar graph showing mass of VAT, subcutaneous adipose tissue (SAT), andspleen in aged control and fTreg KO mice (˜15 months, n=9 per group).FIG. 3D is a linear graph showing cumulative food consumption of controland fTreg KO mice (˜8-9 months old, n=8 per group). FIGS. 3E and 3F arebar graphs showing average 24 hour respiratory exchange ratio (RER) of(FIG. 3E) and average VO₂ (FIG. 3F) consumed by aged control and fTregKO mice (˜11 months, n=6 per group). FIG. 3G is a bar graph showing corebody temperature of control and fTreg mice (˜13 months old, n=9 pergroup). FIG. 3H is a scatter plot showing principal component analysisof non-macrophage adipolmmune Profiles (AIPs) of young (12 weeks), aged(˜15 months) and aged-fTreg KO (˜15 months) mice. Data representsmean±s.e.m. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

FIGS. 4A and 4B are pie charts and a bar graph showing visceral adiposetissue (VAT) adipolmmune Profiles (AIPs) of aged fTreg KO and controlmice. FIG. 4A are pie charts showing adipolmmune Profiles of aged (˜14months old) fTreg KO and control mice male mice depicting immune cellabundance, expressed as percentage of CD45.2⁺ cells. Entirety of immunecompartment (top) was further divided into pan-macrophage (middle) andnon-macrophage (bottom) pie charts (n=9 mice per group). FIG. 4B is abar graph showing immune cell abundance between fTreg KO and controlmice, expressed as cells per gram of VAT (n=9 mice per group). #, falsediscovery rate <2%. Data represents mean±s.e.m.

FIGS. 5A and 5B are two bar graphs showing subcutaneous adipose tissue(SAT) adipoImmune and spleen immune profiles of fTreg KO and controlmice. FIGS. 5A and 5B compare immune cell abundance between fTreg KO andcontrol mice, expressed as cells per gram of subcutaneous adipose tissue(SAT, FIG. 5A) and spleen (FIG. 5B) (n=9 mice per group). Datarepresents mean±s.e.m.

FIGS. 6A-6D are bar graphs and histological images showing that agedfTreg KO mice did not show signs of systemic autoimmunity or breakdownin peripheral tolerance. FIG. 6A is a bar graph showing percentage ofsplenic naive CD4⁺ T cells as defined by CD62^(hi)CD44^(lo) relative tototal CD4⁺ Foxp3-YFP⁻ CD25⁻ population (n=9 mice per group). FIG. 6B areeight representative histology images of gastrointestinaltract—duodenum, jejunum, ileum, and colon (left to right), (n=3 mice pergroup). There were no significant lesions observed or differences ininflammation, epithelial changes, or mucosal architecture between thetwo groups (hematoxylin and eosin (H&E), ×100, scale bar 50 μm). FIG. 6Cis a bar graph comparing the histopathology score in the small intestineand colon of fTreg KO and control mice. The severity and extent ofinflammation and epithelial changes as well as mucosal architecture wereeach graded on a score of 1 (minimal) to 5 (severe) and added to obtainan overall score over 20. There were minimal inflammatory changes withno significant differences between groups. FIG. 6D is a bar graphshowing a multiplex inflammation panel of serum from fTreg KO andcontrol mice (n=4 pooled samples (3 mice per sample) per group).

FIGS. 7A-7P are linear and bar graphs and histological images showingthat loss of fTregs protected against the clinical hallmarks ofage-associated insulin resistance. FIG. 7A is a bar graph showing fTreglevels in visceral adipose tissue (VAT) from control and fTreg KO(Foxp3^(Cre) PPARg^(fl/fl)) mice in young (control n=6; fTreg KO n=6),aged (˜15 months old; control n=10; fTreg KO n=10), and obese mice(control n=6; fTreg KO n=8). FIGS. 7B and 7C are two bar graphs showingfasting serum glucose (FIG. 7B) and insulin levels (FIG. 7C) in controland fTreg KO mice in young (control n=9; fTreg KO n=9), aged (36 weeksold, control n=9; fTreg KO n=11), and obese mice (control n=10; fTreg KOn=10). FIGS. 7D-7F are six linear graphs showing glucose tolerance andinsulin tolerance tests of control and fTreg KO mice at young (FIG. 7D,12 weeks, control n=8; fTreg KO n=8), aged (FIG. 7E, 36-37 weeks,control n=8; fTreg KO n=9), and obese mice (FIG. 7F, control n=9; fTregKO n=10). FIG. 7G shows four representative images of hematoxylin andeosin (H&E) staining of visceral adipose tissue (VAT, gonadal) from ˜14month old control (n=3) and fTreg KO mice (n=5) (scale bar, 50 μM). FIG.7H is a box and whisker plot of adipocyte size distribution in visceraladipose tissue from control (n=3) and fTreg KO mice (n=3) (˜14 monthsold). FIG. 7I is a bar graph comparing ad libitum fed serumnon-esterified fatty acid (NEFA) levels in aged control (n=9) and fTregKO mice (n=10). FIG. 7J is a bar graph comparing serum resistin levelsin ˜14 month old fasted control and fTreg KO mice (n=4 pooled samples (2mice per sample) per group). FIG. 7K is a bar graph comparingpost-prandial glucose uptake in visceral adipose tissue of aged control(n=5) and fTreg KO mice (n=4). FIG. 7L is a bar graph showingrepresentative images of hematoxylin and eosin (H&E) staining of liverfrom ˜14 month old control (n=3) and fTreg KO mice (n=5) (scale bar, 200μM). FIG. 7M is a bar graph comparing hepatic triglyceride levels in ˜14month old control (n=5) and fTreg KO mice (n=3). FIG. 7N is a bar graphshowing fasting serum triglycerides in ˜14 month old control (n=9) andfTreg KO mice (n=10). FIG. 7O is a bar graph showing fTregs, expressedas % of total CD45.2⁺ cells, in control and IL2/anti-IL2 treated mice(n=3 mice per group). FIG. 7P is a bar graph showing relative glucoseuptake in VAT of 16 week old wild-type and IL2/anti-IL2 treated mice(n=4 mice per group). Data represents mean±s.e.m. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001.

FIG. 8 is a bar graph comparing body weights of weight-matched cohortsof control and fTreg KO young, aged, and obese mice. Body weights offTreg KO and control male mice used in weight-matched metabolic studiesin young (12 week old; control n=9; fTreg KO n=9), aged (36 week old;control, n=9 mice; fTreg KO, n=11 mice), and obese (diet-induced obese(DIO), 12 weeks of high fat diet (HFD) starting at 12 weeks old; controln=10; fTreg KO n=10) settings.

FIGS. 9A and 9B are pie charts and a bar graph showing visceral adiposetissue (VAT) adipolmmune profiles of obese fTreg KO and control mice.FIG. 9A are pie charts of adipolmmune profiles of obese (diet-inducedobese (DIO), 16 weeks high fat diet (HFD) started at 12 weeks of age)control (n=6 mice) and fTreg KO (n=8 mice) male mice depicting immunecell abundance, expressed as percentage of CD45.2⁺ cells. Entirety ofimmune compartment (top) was further divided into pan-macrophage(middle) and non-macrophage (bottom) pie charts. FIG. 9B is a bar graphshowing immune cell abundance between fTreg KO and control mice,expressed as cells per gram of VAT (n=9 mice per group). Data representsmean±s.e.m.

FIG. 10 includes two pie charts showing frequency of small, medium, andlarge adipocytes in visceral adipose tissue (VAT) of aged control andfTreg KO mice. Frequency of small (0-5000 μm²), medium (5000-10,000μm²), and large (>10,000 μm²) adipocytes in VAT of aged control andfTreg KO mice (n=3 mice per group, 850 adipocytes counted from controlmice, 269 adipocytes counted from fTreg KO adipose).

FIGS. 11A and 11B depict an RNA-Seq analysis heat map and a scatter plotof fTreg gene expression. FIG. 11A is a heat map showing expression ofseveral canonical Treg genes across fat and splenic Tregs and fat andsplenic Tconv cells. Cells were pooled from 3 and 4 mice beforeisolating RNA for subsequent RNA-Seq analysis. FIG. 11B is a scatterplot comparing expression of ST2 across all hematopoietic cellscatalogued in the Immunological Genome Project database. Position ofadipose CD4⁺ CD25⁺ T cells is marked.

FIGS. 12A-12I are heat maps and graphs showing that fTreg depletionimproved adipose glucose uptake. FIGS. 12A and 12B are a heat map and ascatter plot showing hierarchical clustering of differentially expressedgenes between fat Tregs and Tconv and splenic Tregs and Tconv cells(FIG. 12A), and fold change in expression levels of differentiallyexpressed genes (FIG. 12B) across fat Tregs and Tconv and splenic Tregsand Tconv cells from Foxp3-Thy1.1 mice (47 weeks, cells pooled from 3 to4 mice). Fat Treg cluster genes are labeled in red. Position of ST2 ismarked. FIGS. 12C and 12D are a line graph and a bar graph showingrepresentative FACS analysis (FIG. 12C) and quantification of ST2expression (FIG. 12D) in CD4⁺ T cells from aged mice (45 weeks, n=5mice). FIG. 12E is a bar graph comparing total number of ST2⁺ Tregs andTconv cells per gram of tissue in visceral adipose tissue (VAT) andspleen (n=5 mice). FIG. 12F is a line graph of FACS histograms, FIG. 12Gis a bar graph showing quantification of Tregs (% Foxp3⁺ of CD45⁺ CD4⁺population), and FIG. 12H is a bar graph depicting cells per gram oftissue in VAT and spleen after IL-33 or PBS treatment (16 weeks, n=5mice per group). FIG. 12I is a bar graph showing ex vivo glucose uptakein VAT from wild type mice after control or IL-33 treatment (16 weeks,n=5 mice per group). FIG. 12J is a bar graph showing percentages ofCD4⁺, fTreg, and CD4⁺ Tconv cells in VAT, and FIG. 12K is a bar graphshowing ex vivo insulin stimulated glucose uptake in VAT from wild typemice after anti-ST2 depleting antibody or isotype control treatment (˜45weeks old, n=4 mice per group). FIG. 12L is schematic of an adipo-immunemodel of metabolic aging. Data represents mean±s.e.m. *p<0.05, **p<0.01.

FIGS. 13A-13C are three bar graphs showing that depleting anti-ST2antibody treatment does not promote T cell activation associated withsystemic Treg-dysfunction.

FIG. 13A is a bar graph comparing total weight before beginning courseof anti-ST2 or isotype control antibodies (Day 0) and upon terminalanalysis. FIGS. 13B and 13C are two bar graphs showing spleen weight(FIG. 13B) and percentage of splenic naive CD4⁺ T cells as defined byCD62^(hi) CD44^(lo) relative to total splenic CD45⁺ CD4⁺ CD25⁻ T cellpopulation (FIG. 13C) of mice upon terminal analysis (Day 3, n=4 miceper group).

DETAILED DESCRIPTION OF THE INVENTION

As described herein below, the present invention features compositionsand methods for treating or preventing age-related insulin resistance,type 2 diabetes and related disorders. The method involves depletingfTreg cells with an anti-ST2 antibody to decrease age-related fTregaccumulation and restore insulin sensitivity, thereby treatingage-related insulin resistance, type 2 diabetes and related disorders.

The invention is based, at least in part, on the discovery that adipose(fat)-resident regulatory T cells (fTregs) accumulate in adipose tissueas a function of age, and their accumulation in adipose is a driver ofage-associated insulin resistance. Treatment with anti-ST2 antibodiesdepleted fTregs while preserving splenic Treg numbers. Additionallyadipose from aged mice treated with anti-ST2 antibody had increasedinsulin sensitivity compared to controls.

Age-associated insulin resistance and obesity-associated insulinresistance are two physiologically distinct forms of adult onsetdiabetes. While macrophage-driven inflammation is a core driver ofobesity-associated insulin resistance, the underlying mechanisms of theobesity-independent yet highly prevalent age-associated insulinresistance are largely unexplored. Comparative adipo-immune profilingrevealed that fat-resident regulatory T cells, termed fTregs,progressively accumulated in adipose tissue as a function of age, butnot obesity. Supporting the existence of two distinct mechanismsunderlying age-associated versus obesity-associated insulin resistance,mice deficient in fTregs were protected against age-associated insulinresistance, yet remained susceptible to obesity-associated insulinresistance and metabolic disease. In contrast, selective depletion offTregs via anti-ST2 antibody treatment increased adipose tissue insulinsensitivity. These findings established that distinct immune cellpopulations within adipose tissue underlie aging- and obesity-associatedinsulin resistance and implicated fTregs as adipo-immune drivers andpotential therapeutic targets in the treatment of age-associated insulinresistance.

The immune system is complex, and the relative populations of differentimmune cells within adipose tissue are essentially uncharacterized. Tobetter understand the immune system in aged adipose tissue, aquantitative, global picture of the immune system within adipose tissuewas assembled using AdipoImmune Profile and fluorescence activated cellsorting (FACS).

Fat-resident regulatory T cells, termed fTregs, drove age-associatedinsulin resistance and can be specifically depleted to increase adiposeinsulin sensitivity. Comparative AdipoImmune profiling in young, aged,and obese mice revealed that fTregs progressively enriched in adipose asa function of age, but not obesity. fTreg-deficient mice were protectedfrom age-associated insulin resistance and its accompanyingphysiological hallmarks. In contrast, fTreg-deficiency offered noprotection from obesity-associated insulin resistance. One generalproblem about studying tissue-specific immune cells is that there isgenerally not a clean way of depleting, expanding, or perturbing a givenimmune cell type in a tissue-specific manner. Leveraging the highdifferential expression of ST2 in fTregs compared to splenic Tregs, itwas found that treatment with anti-ST2 antibody depletes fTregs andincreases adipose insulin sensitivity while preserving splenic Tregnumbers. Taken together, it was demonstrated that distinctimmunophysiologies underlie aging-versus obesity-associated insulinresistance and posit fTregs as adipoimmune drivers of and potentialtherapeutic targets against age-associated insulin resistance.Accordingly, the invention provides methods featuring an anti-ST2antibody for enhancing insulin sensitivity in adipose tissue, andtreating age-related insulin resistance, type 2 diabetes and relateddisorders.

ST2 Antibodies

Antibodies that selectively bind ST2 are useful in the methods of theinvention. Methods of preparing antibodies are well known to those ofordinary skill in the science of immunology. As used herein, the term“antibody” means not only intact antibody molecules, but also fragmentsof antibody molecules that retain immunogen-binding ability. Suchfragments are also well known in the art and are regularly employed bothin vitro and in vivo. Accordingly, as used herein, the term “antibody”means not only intact immunoglobulin molecules but also the well-knownactive fragments F(ab′)₂, and Fab. F(ab′)₂, and Fab fragments that lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). The antibodiesof the invention comprise whole native antibodies, bispecificantibodies; chimeric antibodies; Fab, Fab′, single chain V regionfragments (scFv), fusion polypeptides, and unconventional antibodies.

Unconventional antibodies include, but are not limited to, nanobodies,linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062, 1995),single domain antibodies, single chain antibodies, and antibodies havingmultiple valencies (e.g., diabodies, tribodies, tetrabodies, andpentabodies). Nanobodies are the smallest fragments of naturallyoccurring heavy-chain antibodies that have evolved to be fullyfunctional in the absence of a light chain. Nanobodies have the affinityand specificity of conventional antibodies although they are only halfof the size of a single chain Fv fragment. The consequence of thisunique structure, combined with their extreme stability and a highdegree of homology with human antibody frameworks, is that nanobodiescan bind therapeutic targets not accessible to conventional antibodies.Recombinant antibody fragments with multiple valencies provide highbinding avidity and unique targeting specificity to cancer cells. Thesemultimeric scFvs (e.g., diabodies, tetrabodies) offer an improvementover the parent antibody since small molecules of ˜60-100 kDa in sizeprovide faster blood clearance and rapid tissue uptake See Power et al.,(Generation of recombinant multimeric antibody fragments for tumordiagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu etal. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumortargeting and imaging. Tumor Targeting, 4, 47-58, 1999).

Various techniques for making and unconventional antibodies have beendescribed. Bispecific antibodies produced using leucine zippers aredescribed by Kostelny et al. (J. Immunol. 148(5):1547-1553, 1992).Diabody technology is described by Hollinger et al. (Proc. Natl. Acad.Sci. USA 90:6444-6448, 1993). Another strategy for making bispecificantibody fragments by the use of single-chain Fv (sFv) diners isdescribed by Gruber et al. (J. Immunol. 152:5368, 1994). Trispecificantibodies are described by Tutt et al. (J. Immunol. 147:60, 1991).Single chain Fv polypeptide antibodies include a covalently linkedVH::VL heterodimer which can be expressed from a nucleic acid includingV_(H)- and V_(L)-encoding sequences either joined directly or joined bya peptide-encoding linker as described by Huston, et al. (Proc. Nat.Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Pat. Nos.5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos.20050196754 and 20050196754.

In one embodiment, an antibody that binds ST2 is monoclonal.Alternatively, the anti-ST2 antibody is a polyclonal antibody. Thepreparation and use of polyclonal antibodies are also known the skilledartisan. The invention also encompasses hybrid antibodies, in which onepair of heavy and light chains is obtained from a first antibody, whilethe other pair of heavy and light chains is obtained from a differentsecond antibody. Such hybrids may also be formed using humanized heavyand light chains. Such antibodies are often referred to as “chimeric”antibodies.

In general, intact antibodies are said to contain “Fc” and “Fab”regions. The Fc regions are involved in complement activation and arenot involved in antigen binding. An antibody from which the Fc′ regionhas been enzymatically cleaved, or which has been produced without theFc′ region, designated an “F(ab¢)₂” fragment, retains both of theantigen binding sites of the intact antibody. Similarly, an antibodyfrom which the Fc region has been enzymatically cleaved, or which hasbeen produced without the Fc region, designated an “Fab′” fragment,retains one of the antigen binding sites of the intact antibody. Fab¢fragments consist of a covalently bound antibody light chain and aportion of the antibody heavy chain, denoted “Fd.” The Fd fragments arethe major determinants of antibody specificity (a single Fd fragment maybe associated with up to ten different light chains without alteringantibody specificity). Isolated Fd fragments retain the ability tospecifically bind to immunogenic epitopes.

Antibodies can be made by any of the methods known in the art utilizingST2 polypeptide, or immunogenic fragments thereof, as an immunogen. Onemethod of obtaining antibodies is to immunize suitable host animals withan immunogen and to follow standard procedures for polyclonal ormonoclonal antibody production. The immunogen will facilitatepresentation of the immunogen on the cell surface. Immunization of asuitable host can be carried out in a number of ways. Nucleic acidsequences encoding an ST2 polypeptide or immunogenic fragments thereof,can be provided to the host in a delivery vehicle that is taken up byimmune cells of the host. The cells will in turn express the receptor onthe cell surface generating an immunogenic response in the host.Alternatively, nucleic acid sequences encoding a ST2 polypeptide, orimmunogenic fragments thereof, can be expressed in cells in vitro,followed by isolation of the receptor and administration of the receptorto a suitable host in which antibodies are raised.

Alternatively, antibodies against an ST2 polypeptide may, if desired, bederived from an antibody phage display library. A bacteriophage iscapable of infecting and reproducing within bacteria, which can beengineered, when combined with human antibody genes, to display humanantibody proteins. Phage display is the process by which the phage ismade to ‘display’ the human antibody proteins on its surface. Genes fromthe human antibody gene libraries are inserted into a population ofphage. Each phage carries the genes for a different antibody and thusdisplays a different antibody on its surface.

Antibodies made by any method known in the art can then be purified fromthe host. Antibody purification methods may include salt precipitation(for example, with ammonium sulfate), ion exchange chromatography (forexample, on a cationic or anionic exchange column preferably run atneutral pH and eluted with step gradients of increasing ionic strength),gel filtration chromatography (including gel filtration HPLC), andchromatography on affinity resins such as protein A, protein G,hydroxyapatite, and anti-immunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineeredto express the antibody. Methods of making hybridomas are well known inthe art. The hybridoma cells can be cultured in a suitable medium, andspent medium can be used as an antibody source. Polynucleotides encodingthe antibody of interest can in turn be obtained from the hybridoma thatproduces the antibody, and then the antibody may be producedsynthetically or recombinantly from these DNA sequences. For theproduction of large amounts of antibody, it is generally more convenientto obtain an ascites fluid. The method of raising ascites generallycomprises injecting hybridoma cells into an immunologically naivehistocompatible or immunotolerant mammal, especially a mouse. The mammalmay be primed for ascites production by prior administration of asuitable composition (e.g., Pristane).

Monoclonal antibodies (Mabs) produced by methods of the invention can be“humanized” by methods known in the art. “Humanized” antibodies areantibodies in which at least part of the sequence has been altered fromits initial form to render it more like human immunoglobulins.Techniques to humanize antibodies are particularly useful when non-humananimal (e.g., murine) antibodies are generated. Examples of methods forhumanizing a murine antibody are provided in U.S. Pat. Nos. 4,816,567,5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.

Diagnostics

The present invention features diagnostic assays for the detection ofage-related insulin resistance, type 2 diabetes and related disorders orthe propensity to develop such conditions. In one embodiment, levels ofST2 polypeptides and/or polynucleotides are measured in a subject sample(e.g., adipose tissue, fTregs) and used as an indicator of age-relatedinsulin resistance, type 2 diabetes and related disorders or thepropensity to develop such conditions. In another embodiment, the numberof fTregs are measured in a subject sample (e.g., adipose tissue) andused as an indicator of age-related insulin resistance, type 2 diabetesand related disorders or the propensity to develop such conditions.Detection of an increase in an ST2 polypeptide or polynucleotideexpression in an fTreg or detecting an increase in the number of fTregsin the sample relative to a control is indicative of age-related insulinresistance, type 2 diabetes and related disorders or the propensity todevelop such conditions.

Standard methods may be used to measure levels of ST2 polypeptidesand/or polynucleotides in fTregs. Such methods include immunoassay,ELISA, western blotting using an anti-ST2 antibody, andradioimmunoassay. Methods for measuring the number of fTregs present ina sample are known in the art and described herein below (e.g., usingadipose immune profiling).

Diagnostic Kits

The invention also provides for a diagnostic test kit that comprises anantibody or other means for detecting an ST2 polypeptide. Desirably, thekits includes instructions for the use of the kit in the methods of theinvention. In one embodiment, the kit further comprises reagents,equipment (test tubes, reaction vessels, needles, syringes, etc.). Theinstructions provided in a kit according to the invention may bedirected to suitable operational parameters in the form of a label or aseparate insert. Optionally, the kit may further comprise a standard orcontrol information so that the test sample can be compared with thecontrol information standard to determine whether a consistent result isachieved.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1: Age-Associated Insulin Resistance was Driven byfTregs

The young, lean state is associated with insulin sensitivity, howeverboth aging and obesity can lead to the development of insulin resistance(FIG. 1A). To distinguish key immune cell types that drive age-versusobesity-associated insulin resistance, the immune cell components ofadipose depots were quantitatively profiled using a flow cytometryapproach termed adipo-immune profiling (AIP) (FIGS. 2A-C, Table 1). Incontrast to the decrease in anti-inflammatory M2 adipose tissuemacrophages (ATMs) and eosinophils observed in obesity-driven insulinresistance, AIP revealed that these cell populations were largelyunperturbed in visceral adipose tissue from aged mice (M2 ATMs—aged:33.6±3.8%, young: 29.8±4.1%, obese: 22.9±6.3%; eosinophils—aged:4.4%±1.6%, young: 4.7%±0.7%, obese: 0.8%±1.0%, FIG. 1B). Rather, therelative portion of the non-macrophage compartment was significantlyincreased in aged compared to young or obese mice (aged: 24.3±4.6%,young: 17.9±2.8%, obese 15.7±3.8%, FIG. 1B), which was largelyattributable to a ˜13.5 fold expansion in the fat-resident regulatory Tcell (fTreg) population (aged: 5.0±1.2%, young: 0.4±0.1%, obese:0.1±0.1%, FIGS. 1B and 1C). These varying AIP signatures of adiposetissue indicated that distinct pathophysiologic processes drive age- andobesity-associated insulin resistance and implicated fTregs as potentialdrivers of age-associated insulin resistance.

TABLE 1 Antibodies used to molecularly identify the given immune celltype. Immune Cell Type Molecular Identification Scheme TCRγδ CD45.2⁺F4/80⁻ CD3⁺ TCRb⁻ TCRγ⁺ CD8⁺ CD45.2⁺ F4/80⁻ CD3⁺ TCRb⁺ CD4⁻ CD8⁺ TregCD4⁺ CD45.2⁺ CD4⁺ CD25⁺ Foxp3⁺ Naive CD4⁺ CD45.2⁺ CD4⁺ CD25⁻ Foxp3⁻CD62L^(hi) CD44^(lo) Activated CD4⁺ CD45.2⁺ CD4⁺ CD25⁻ Foxp3⁻ CD62L^(lo)CD44^(hi) NKT CD45.2⁺ NK1.1⁺ TCRb⁺ NK CD45.2⁺ NK1.1⁺ TCRb⁻ B CD45.2⁺NK1.1⁻ CD19⁺ Eosinophil CD45.2⁺ F4/80⁺ Siglec-F⁺ Neutrophil CD45.2⁺F4/80⁻ CD11c− CD11b⁺ Ly6G⁺ M2 ATM CD45.2⁺ F4/80⁺ CD11c^(med) CD206⁺ M1ATM CD45.2⁺ F4/80⁺ CD11c^(hi) CD206⁻ DN (Double-negative) CD45.2⁺ F4/80⁺CD11c⁻ CD206⁻ ATM

Example 2: Adipolmmune Profiles of Control and fTreg KO Mice LookedNearly Identical

To explore the role of fat-resident regulatory T cells (fTregs) inage-associated insulin resistance, Foxp3^(Cre) Pparg^(fl/fl) mice wereutilized in which Tregs were selectively depleted from visceral adiposetissue (fTreg KO mice, FIG. 3A, FIGS. 4A and 4B, Cipoletta et al.,Nature. doi:10.1038/nature11132, 2012) without significantly alteringthe immune profiles of subcutaneous adipose tissue or spleen (FIGS. 5Aand 5B). Importantly, the visceral adipose tissue-specific loss offTregs did not elicit any overt signs of systemic inflammation generallyassociated with Treg dysfunction. Aged fTreg KO mice had normal-sizedspleens and increased CD62L^(hi) CD44^(lo) naive CD4⁺ T cell populationscompared to wild-type controls (FIG. 3C, FIG. 6A). The normal intestinalhistology provided additional evidence that the Treg population was notperturbed (FIGS. 6B and 6C). Furthermore, no differences were observedin the levels of inflammatory cytokines, including TNFα, IL1b, IL6,IFNγ, and IL17, in the serum of aged fTreg KO compared to control mice(FIG. 6D).

Example 3: Meg KO Mice were Protected Against Age-Associated, but notObesity-Associated Insulin Resistance

Importantly, the selective loss of fTregs attenuated many of thehallmarks of age-associated metabolic dysregulation. They weighed lessthan control mice and were leaner (decreased visceral adipose tissue andsubcutaneous adipose tissue adiposity) despite increased foodconsumption (FIGS. 3B-3D). In addition, the respiratory exchange ratio(RER, FIG. 3E), oxygen consumption (FIG. 3F), and core body temperature(FIG. 3G) were increased in aged fTreg KO mice relative to control mice.These marked improvements indicated that the age-associated metabolicphenotype was closely linked with visceral adipose tissue immuneresponses, and that in the aged setting, a reduction in fTreg numbersmight be protective. Indeed, the adipo-immune profilings of aged fTregKO mice were shifted towards those of young mice, as visualized byprincipal component analysis (FIG. 3H).

The fTreg KO phenotype was most pronounced in aged mice, though areduction in fTreg levels was also seen in obese fTreg KO mice (FIG.7A). Consistent with the notion that fTregs are drivers ofage-associated metabolic dysregulation, fasting serum glucose andinsulin levels were significantly reduced in aged fTreg KO mice (FIGS.7B and 7C and FIG. 8). Furthermore, aged fTreg KO mice displayed smallerglucose excursions during glucose tolerance tests and increasedsensitivity during insulin tolerance tests compared to weight-matchedcontrol mice (FIG. 7E). Notably, these improvements in glucosehomeostasis were observed only in aged mice; no significant differenceswere seen in young or obese fTreg KO mice (FIGS. 7D and 7F), which wasconsistent with the largely unchanged adipo-immune profiling (AIP) ofobese fTreg KO mice (FIGS. 9A and 9B).

Example 4: Hallmarks of Insulin Resistance were Attenuated in fTreg KOMice Compared to Age-Matched Controls

Histologically, aged fat-resident regulatory T cell (fTreg) KO visceraladipose tissue depots appeared similar to control mice, and inflammatoryprocesses such as crowning were observed at comparable frequencies (FIG.7G). However, adipocytes from aged fTreg KO mice were smaller than thosein control mice (fTreg KO 70% <5000 mm², control ˜41% <5000 mm², FIG. 7Hand FIG. 10), and serum non-esterified free fatty acid (NEFA) levelswere reduced to almost half those of control mice; both indicators ofimproved insulin sensitivity (FIG. 7I). In addition, circulating levelsof the adipokine resistin, which positively correlates with murineinsulin resistance, were reduced in the aged fTreg KO mice (FIG. 7J).Furthermore, aged fTreg KO mice presented with decreased hepaticsteatosis, as determined histologically and by decreased fasting hepaticand serum triglyceride content (FIGS. 7L-7N). In combination, thesefindings indicated that the loss of fTregs in adipose tissue alleviatedmany of the indications of age-associated insulin resistance in mice, aprimary clinical manifestation of metabolic aging.

To more directly associate fTregs with age-associated insulinresistance, basal glucose uptake was measured in adipose tissue ex vivo.Notably, visceral adipose tissue from fTreg KO mice took up almost twicethe amount of glucose as control tissue (FIG. 7K). Conversely, expansionof fTregs in wild-type mice via treatment with IL-2/IL-2 mAb complextreatment abrogated basal glucose uptake in visceral adipose tissue by˜50% (FIGS. 7O and 7P). This inverse correlation between fTreg numbersand glucose uptake in adipose tissue supported a causal associationbetween fTregs and insulin resistance during aging.

Example 5: fTregs are Functionally Distinct from Splenic Tregs and MoreHighly Express the IL-33 Receptor ST2 than Splenic Tregs or ConventionalCD4⁺ T (Tconv) Cells

These findings of an association between fat-resident regulatory T cells(fTregs) and age-associated insulin resistance and metabolic agingindicated that these cells were functionally distinct from splenicTregs. To investigate this notion, the transcriptomes of Tregs werecompared, as well as conventional CD4⁺ T cells, isolated from visceraladipose tissue and spleen. Comparative analyses revealed that whilecertain canonical genes were similarly expressed (e.g. Foxp3, Ctla4, andTigit), visceral adipose tissue and splenic Tregs had discreteexpression signatures, consistent with the indicated functionaldistinction. In particular, Pparg, Gata3, and Irf4 were selectivelyenriched in visceral adipose tissue, but not splenic Tregs (FIG. 11A).Furthermore, unbiased comparative gene expression analyses combined withhierarchical clustering defined extensive fat- and splenic-residenceclusters (1142 genes and 1431 genes, respectively) relative to muchsmaller Pan-Treg Clusters 1 and 2 (56 and 162 genes, respectively).Transcriptionally, fTregs clustered more closely with fat conventionalCD4⁺ T cells than splenic Tregs (FIG. 12A), indicating that thefunctional specification of fTregs was informed by their anatomicallocation within adipose tissue, as well as the expression of the Treglineage-specifying transcription factor Foxp3. It was posited that thetranscriptional differences between fTregs and splenic Tregs (found inthe fTreg cluster of 1049 genes) might provide a therapeutic avenue toselectively manipulate fTreg populations. The IL-33 receptor ST2, whichlies within the fTreg cluster, had been recently implicated in effectorTreg and in particular fTreg development (Vasanthakumar et al., NatImmunol 1-12. doi:10.1038/ni.3085), 2015; Schiering, C. et al., Nature513, 564-568, 2014). Indeed, ST2 was ˜60 and ˜30 times more highlyexpressed in fTregs compared to splenic Tregs and fat conventional CD4⁺T cells, respectively, consistent with the ImmGen database (website ofthe Immunological Genome Project), (FIG. 12B and FIG. 11B). Flowcytometry confirmed that ST2 was expressed on the cell surface of themajority of fTregs, but on relatively few fat conventional CD4⁺ T cells,or splenic Tregs or conventional CD4⁺ T cells (FIGS. 12C and 12D).Furthermore, visceral adipose tissue had ˜10× more ST2⁺ fTregs than ST2⁺fat conventional CD4⁺ T; a similar ratio was seen in the spleen (FIG.12E).

Example 6: Acute Treatment with an Anti-ST2 Antibody LED to PartialDepletion of fTregs Concomitantly with a Reduction in Fat CD4⁺ T (Tconv)Cells and an Increase in Insulin Sensitivity

To explore the therapeutic potential of the IL-33/ST2 signaling pathway,aged mice were initially injected with IL-33 (0.5 mg intraperitoneally(i.p.) on days 0, 2, 4) to expand the fat-resident regulatory T cell(fTreg) population (FIGS. 12F-12H). In agreement with fTreg expansiondriven by IL2/anti-IL2 treatment, mice injected with IL-33 displayedsigns of insulin resistance (basal glucose uptake in visceral adiposetissue reduced to ˜60% of control mice, FIG. 12I). In the converseapproach, acute treatment with an anti-ST2 antibody (200 mg/mouse i.p.on days 0 and 2) was able to partially deplete fTregs (FIG. 12J). Thisdepletion occurred concomitantly with a reduction in fat CD4⁺ T (Tconv)cells, indicating a possible role for ST2 and/or fTregs in maintainingthis population. Importantly, partial depletion of fTregs achieved withacute anti-ST2 treatment was sufficient to increase insulin-stimulatedglucose uptake in visceral adipose tissue (˜25% increase in glucoseuptake compared to control treated mice, FIG. 12K). Furthermore, thisincrease in insulin sensitivity was achieved without any signs ofconventional CD4⁺ T cell activation associated with systemic Tregdysfunction (FIG. 13).

Taken together, these data provide evidence that distinct adipo-immunepopulations drive age- and obesity-associated insulin resistance. Thefindings that fTregs accumulated in mouse adipose tissue as a functionof age and exacerbated the decline of adipose metabolic functionassociated with aging (FIG. 12L), complements the established role of M1ATMs in the decline of adipose metabolic function in the setting ofobesity. Thus, these studies showcased the ability of the immunecompartment within adipose to drive key aspects of metabolic aging, inparticular insulin resistance. Given the classical immune suppressiveand anti-inflammatory nature of Tregs, it was speculated that thechronic inflammatory processes that drove obesity-associated insulinresistance were likely not driving age-associated insulin resistance.Indeed, it was possible that maintaining a certain degree ofinflammation was beneficial for adipose tissue remodeling and itsmetabolic function, and the increased abundance of fTregs in the agedstate might prevent beneficial inflammatory processes like adiposeremodeling necessary for maintenance of adipose insulin sensitivity.Finally, this study indicated that selective targeting of fTregs mightrepresent a new therapeutic avenue in the treatment of age-relateddiabetes, and more generally provided a proof-of-principle thatdifferent subtypes of Tregs could be targeted in a clinically achievablemanner bringing researchers closer to the promise that theidentification of Tregs heralded many years ago.

The experiments above were performed with the following methods andmaterials.

Methods and Materials Mice

All mice were bred or housed in specific pathogen-free facilities at TheSalk Institute for Biological Studies or purchased from TaconicBiosciences. C57BL/6NTac and 129S6/SvEvTac mice were purchased fromTaconic Biosciences for comparative adipo-immune profiling (AIP).Age-matched retired breeders were purchased for AIP of aged adipose, andDIO C57BL/6NTac mice were purchased for profiling of obese adipose.fTreg KO mice were generated by crossingB6.129(Cg)-Foxp3^(tm4(YFP/cre)Ayr)/J (Rubtsov, Y. P. et al., Immunity28, 546-558, 2008) and B6.129-Pparg^(tm2Rev)/J (He et al., PNAS 100,15712-15717, 2003) mice. The Foxp3^(Thy1.1) reporter mice were utilizedwhen isolating Tregs and conventional CD4⁺ T cells from spleen and fatfor subsequent RNA-Seq analysis. Mice within The Salk Institute forBiological Studies received autoclaved normal chow (MI laboratory rodentdiet 5001, Harlan Teklad) or autoclaved HFD (60 kcal % fat, ResearchDiets). All mice used for studies were male.

Adipo-Immune Profiling (AIP)

Visceral (epididymal) and subcutaneous (inguinal) adipose depots weredissected from mice after 10 mL PBS perfusion through left ventricle.Inguinal lymph nodes resident in inguinal adipose were removed. Adiposewas minced into fine pieces (2-5 mm³) and digested in adipocyteisolation buffer (100 mM HEPES pH7.4, 120 mM NaCl, 50 mM KCl, 5 mMglucose, 1 mM CaCl2, 1.5% BSA) containing 1 mg/ml collagenase at 37° C.with intermittent shaking for 1.5 hours. The suspension was then passedthrough a 100 μm mesh to remove undigested clumps and debri. Theflowthrough was allowed to stand for 10 minutes to separate the floatingadipocyte fraction and infranatant containing the stromal vascularfraction. The infranatant was removed while minimally disturbing thefloating adipocyte fraction and centrifuged at 400 g for 10 minutes. Thepellet containing the stromal vascular fraction was washed once in 10 mLRPMI. The resultant isolated cells were subjected to FluorescenceActivated Cell Sorting (FACS) analysis. The following antibodies wereused to assemble the Adipo-immune Profile with the manufacturerpreceding and clone number within parantheses: BioLegend—CD45.2 (104),CD44 (IM7), CD62L (MEL-14), TCRg/d (GL3), CD19 (6D5), CD25 (PC61), CD206(C068C2), CD301 (LOM-14); eBioscience—CD3 (145-2C11), CD25 (PC61), CD4(RM4-5), TCRb (H57-597), B220 (RA3-6B2), NK1.1 (PK136), CD49b (DX5),Foxp3 (FJK-16s), F4/80 (BM8), CD11c (N418), CD11b (M1/70); Tonbobiosciences—F4/80 (BM8.1), CD4 (RM4-5), CD44 (IM7), CD62L (MEL-14), Ly6G(RB6-8C5); BD Pharmingen—Siglec-F (E50-2440); BD Biosciences—CD8a(53-6.7). When analyzing myeloid cell populations, Fc blocking antibody(CD16/CD32, Tonbo biosciences, 2.4G2) was utilized. Cells were analyzedusing the BD® FACS Aria instrument and FlowJo software.

Body Composition and Adipocyte Size Analyses

Body composition was measured with an ECHO® MRI-100 body compositionanalyzer (ECHO MEDICAL SYSTEMS®). Visceral adipose tissue (VAT)(epididymal adipose) was dissected, and the wet weight was determined.Adipose tissues were fixed in 10% formalin, sectioned, and stained inhematoxylin and eosin. An adipocyte cross-sectional area was determinedfrom photomicrographs of VAT using ImageJ.

In Vivo Metabolic Phenotype Analysis

Real-time metabolic analyses were conducted in a Comprehensive LabAnimal Monitoring System (COLUMBUS INSTRUMENTS®). CO₂ production, 02consumption, and ambulatory counts were determined for at least threeconsecutive days and nights after at least 24 hours for adaptationbefore data recording.

Principal Component Analysis (PCA) of Adipo-Immune Profiling (AIP)

Non-macrophage immune cell populations, described as percent of thetotal CD45.2⁺ immune compartment, were inputted into MetaboAnalyst 3.0(a comprehensive tool suite for metabolomic data analysis) for principalcomponent analysis (PCA). No normalizations, transformations, orscalings were implemented.

Glucose Homeostasis Studies

Fasting was induced for 6 hours, except for glucose tolerance tests(GTTs), which were conducted after overnight fasting. Glucose (1-2 g/kg,intraperitoneally (i.p.)) and insulin (0.5-1.0 U/kg, i.p.) was injectedfor GTTs and insulin tolerance tests (ITTs), respectively. Blood glucosewas monitored using a Nova Max Plus glucometer.

Histological Analyses

Sections (4 mm) of fixed tissues were stained with haematoxylin andeosin according to standard procedures. Histopathological scores weregraded on blinded samples for severity and extent of inflammation andmorphological changes by a pathologist.

Serum Analyses

Blood was collected by tail bleeding or right atrial puncture.Non-esterified fatty acids (Wako) and triglycerides (Thermo) weremeasured using colorimetric methods. Serum insulin levels (ULTRASENSITIVE INSULIN™, CRYSTAL CHEM®) were measured by ELISAs. Serumcytokine and metabolic hormone levels were analyzed by the LUMINEX™BIO-PLEX® system using the Mouse Cytokine 23-Plex Panel and DiabetesPanel, respectively, as according to the manufacturer'sinstructions)(BIO-RAD®.

Core Body Temperature

Mice were single housed, and core body temperature was measured with aclinical rectal thermometer (THERMALERT™ model TH-5; PHYSITEMP®) at 1:30PM. The probe was dipped in a room temperature lubricating glycerolbefore insertion.

Ex Vivo 2-DG Uptake Assay

Adipose was dissected from mouse, cut into small pieces with scissors,washed and incubated for 30 minutes with Krebs-Ringer Bicarbonate HEPESbuffer (KRBH, 120 mM NaCl, 4 mM KH₂PO₄, 1 mM MgSO₄, 0.75 mM CaCl₂, 30 mMHepes, 10 mM NaHCO₃, pH 7.4, supplemented with 1% fatty-acid free BSA).For determination of exogenous insulin-stimulated 2-deoxy-D-glucose(2-DG) uptake, adipose was incubated in KRBH with 100-200 nM insulin for20 minutes in 37° C. Cold 2-DG and hot 2-DG-1.2-³H(N) was added toincubated adipose such that the final concentration of cold 2-DG was 0.1mM and final quantity of hot 2-DG-1.2-³H(N) was 0.1 μCi (assuming totalreaction volume ˜400 uL). Adipose was further incubated 20 minutes in37° C., then washed three times with PBS before being lysed byscintillation fluid. 2-DG uptake was determined by measuringscintillation counts normalized to adipose mass utilized for assay.Non-specific 2-DG uptake levels were determined by treating adipose withcytochalasin B (0.1 μM final concentration) before addition of cold andhot 2-DG.

IL-2-Anti-IL-2 Complex and IL-33 Injections

IL-2-anti-IL-2 complexes were prepared by incubating 2 μg of murine IL-2(Biolegend) with 10 μg of anti-IL-2 antibody (JES6.1, BIOXCELL®) in atotal volume of 200 μL of PBS for 30 minutes at 37° C. (amounts givenper injection). Mice were injected intraperitoneally (i.p.) three times(days 0, 1, 2) and analyzed on day 8. For IL-33 expansion assays, micewere injected i.p. with 0.5 μg of recombinant murine IL-33 in PBS (R&DSYSTEMS®) three times (days 0, 2, 4) and analyzed on day 6. PBS was usedfor control injections.

RNA-Seq Library Generation

Total RNA was isolated from sorted cells using TRIZOL® reagent(IINVITROGEN®) as per the manufacturer's instructions and treated withDNASEI® (QIAGEN®) for 30 minutes at 22° C. Sequencing libraries wereprepared from 10-100 ng of total RNA using the TRUSEQ® RNA samplepreparation kit v2 (ILLUMINA®) according to the manufacturer's protocol.Briefly, mRNA was purified, fragmented and used for first- andsecond-strand cDNA synthesis followed by adenylation of 3′ ends. Sampleswere ligated to unique adaptors and subjected to PCR amplification.Libraries were then validated using the 2100 BIOANALYZER® (AGILENT®),normalized and pooled for sequencing. RNA-Seq libraries prepared fromtwo biological replicates for each experimental condition were sequencedon the ILLUMINA® HISEQ® 2500 using barcoded multiplexing and a 100-bpread length.

High-Throughput Sequencing and Analysis

Image analysis and base calling were done with ILLUMINA® CASAVA®-1.8.2.This yielded a median of 29.9M usable reads per sample. Short readsequences were mapped to a UCSC mm9 reference sequence using the RNA-Seqaligner STAR® (Dobin, A. et al., Bioinformatics 29, 15-21, 2012). Knownsplice junctions from mm9 were supplied to the aligner and de novojunction discovery was also permitted. Differential gene expressionanalysis, statistical testing and annotation were performed usingCUFFDIFF® 2 (Trapnell et al., Nat Biotechnol 31, 46-53, 2012).Transcript expression was calculated as gene-level relative abundance infragments per kilobase of exon model per million mapped fragments andemployed correction for transcript abundance bias (Roberts et al.,Bioinformatics 27, 2325-2329, 2011). RNA-Seq results for genes ofinterest were also explored visually using the UCSC Genome Browser.

Hierarchical Clustering

Differentially expressed gene names and corresponding fragments perkilobase of exon per million fragments mapped (FPKM) values acrosssamples were inputted into GENE-E (Broad Institute) for hierarchicalclustering analysis (implemented one minus pearson correlation forsample and gene distance metrics and the average linkage method) andvisualization. Gene cluster names were created to describe the geneexpression characteristics within each cluster (i.e. Fat-ResidenceCluster refers to the gene cluster whose genes were expressed at greaterlevels in T cells residing in fat. Fat-Treg Cluster refers to the genecluster whose genes were expressed highest in only the fTregs.)

ST2 Studies and Anti-ST2 Depleting Antibody Treatment

Fluorescence Activated Cell Sorting (FACS) antibody for ST2 waspurchased from MD BIOPRODUCTS®, clone DJ8. Mice were injectedintraperitoneally (i.p.) with 200 μg depleting anti-ST2 antibodies(Monticelli et al., Nat Immunol 12, 1045-1054, 2011; R&D® systems, clone245707) or isotype control (BIOXCELL®) twice (days 0, 2) and sacrificedfor analysis on day 3.

Statistical Analyses

Statistical analyses were performed with Prism 6.0 (GraphPad). p valueswere calculated using two-tailed unpaired Student's t test. Whenanalyzing adipo-immune profiles, a false discovery rate approach wasutilized to avoid the problem of an inflated false-positive rate due tothe substantial number of hypothesis tests.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A method of enhancing insulin sensitivity or reducing insulinresistance in a cell or tissue, the method comprising contacting thecell or tissue with an anti-ST2 antibody or antigen binding fragmentthereof, thereby decreasing the number of adipose-resident regulatory Tcells (fTreg) and enhancing insulin sensitivity or reducing insulinresistance relative to a reference.
 2. (canceled)
 3. The method of claim1, wherein the cell is an adipocyte or wherein the tissue is adiposetissue.
 4. (canceled)
 5. A method of enhancing insulin sensitivity orreducing insulin resistance in a subject, the method comprisingadministering to the subject an anti-ST2 antibody or an antigen bindingfragment thereof, thereby decreasing the number of adipose-residentregulatory T cells in the subject and enhancing insulin sensitivity orreducing insulin resistance relative to a reference.
 6. (canceled)
 7. Amethod for treating or preventing age-related insulin resistance, type 2diabetes, metabolic dysregulation and related disorders in a subject,the method comprising administering to the subject an anti-ST2 antibodyor antigen binding fragment thereof, thereby treating or preventingage-related insulin resistance, type 2 diabetes, metabolic dysregulationand related disorders in the subject.
 8. (canceled)
 9. The method ofclaim 1, wherein the subject is fifty years old or older. 10.-11.(canceled)
 12. The method of claim 5, wherein the method reduces one ormore of body weight relative to a reference, fasting serum glucoserelative to a reference, or insulin levels relative to a reference. 13.The method of claim 5, wherein the method decreases visceral adiposetissue and/or subcutaneous adipose tissue adiposity relative to areference.
 14. The method of claim 5, wherein the method increasesrespiratory exchange ratio, oxygen consumption, and/or core bodytemperature relative to a reference.
 15. (canceled)
 16. The method ofclaim 5, wherein the method reduces glucose excursions during a glucosetolerance test and/or increases sensitivity during an insulin tolerancetest relative to a reference.
 17. The method of claim 5, wherein themethod reduces serum non-esterified free fatty acid (NEFA) levelsrelative to a reference.
 18. The method of claim 5, wherein the methodincreases insulin sensitivity relative to a reference.
 19. The method ofclaim 5, wherein the method decreases one or more of hepatic steatosis,fasting hepatic and/or serum triglyceride content relative to areference.
 20. (canceled)
 21. The method of claim 1, wherein the methoddoes not produce an autoimmune syndrome.
 22. The method of claim 1,wherein the method specifically decreases fTreg numbers while preservingsplenic Treg numbers.
 23. The method of claim 1, wherein the methodincreases the glucose uptake capacity of visceral adipose tissuecompared to a reference.
 24. (canceled)
 25. A method for identifying asubject having or at risk of developing age-related insulin resistance,type 2 diabetes and related disorders, the method comprising detectingan increase in an ST2 polypeptide or polynucleotide expression in anfTreg or detecting an increase in the number of fTregs relative to acontrol.
 26. The method of claim 25, wherein the fTreg is present in atissue biopsy.
 27. (canceled)
 28. The method of claim 25, wherein theST2 polypeptide is detected in an immunoassay.
 29. A method of reducinginsulin resistance in a subject who has or who is at risk of developingage-related insulin resistance, the method comprising administering tothe subject an anti-ST2 antibody or antigen binding fragment thereof,wherein the subject is identified as having an increase in an ST2polypeptide or polynucleotide expression in an fTreg or detecting anincrease in the number of fTregs relative to a control.
 30. (canceled)31. A kit for treating or preventing age-related insulin resistance, thekit comprising an effective amount of an ST2 antibody in apharmaceutical excipient.